The proposed research focuses on elucidating the putative roles of invasion plasmid antigen D (IpaD) in cellular invasion and intercellular spread of the bacterial pathogen Shigella flexneri, the causative agent of bacillary dysentery. S. flexneri along with numerous other important pathogens such as Yersinia pestis (plague), Salmonella (gastroenteritis), and Pseudomonas aeruginosa (lung infection) utilize the type III secretion system (TTSS) as a means of subverting the normal functions of human cells. For Shigella, IpaD is located at the exposed pole of the TTSS where it senses host cell contact and helps to recruit downstream effector proteins for injection into the membrane and cytoplasm of the host cell to promote bacterial invasion. We recently found evidence that IpaD may not only reside on the surface of S. flexneri, but it is injected into the host cell cytoplasm where it may be localized to gap junctions. Because IpaD may have a role in post-invasion events (i.e., direct cell-to-cell spread), it represents an as yet undescribed aspect of Shigella TTSS function that might be a target for disease prevention. In order to better understand this role of IpaD and its homologues from other systems, a series of experiments have been designed to: a) determine the molecular basis for IpaD's intracellular localization (possibly with gap junctions) in cultured human epithelial cells;b) determine the structural features of IpaD that are responsible for its ability to be recruited to specific sites within host cells;and c) test the hypothesis that interactions between IpaD and host cell proteins influences the efficiency of Shigella intercellular spread. Specifically, fluorescence co-localization, FRET, and affinity chromatography experiments will be utilized in order to both map the intracellular distribution of IpaD and identify intracellular host proteins with which it interacts. Once these proteins are identified, mutation analyses in which small deletions and point mutations in IpaD will be introduced to allow for the mapping of regions necessary for native intracellular localization and specific interaction with cellular proteins. Finally, the ability of both native and mutated forms of IpaD to form plaques in HeLa cell monolayers expressing various gap junction proteins (e.g. connexins) will be done to allow for a more precise understanding of IpaD's possible role as an effector. PUBLIC HEALTH RELEVANCE: The proposed research explores the role of IpaD in the ability of bacterial pathogens, such as Shigella, to invade and spread throughout epithelial cell monolayers. By determining the specific mechanisms and interactions that are responsible for these processes, it will be possible to tailor specific anti-infective treatments to prevent these infections as an alternative to standard antibiotic use.